Project Summary The ability of cells and organisms to maintain protein homeostasis is essential for sustaining viability and health. To ensure the proper folding, sorting and degradation of proteins, cells maintain a robust array of factors termed the proteostasis network. The accumulation of misfolding proteins and intracellular aggregates is a hallmark of a variety of neurodegenerative diseases, including Huntington's disease, Parkinson's disease, and Alzheimer's disease. Originally, this accumulation was thought to occur when protein misfolding outpaced the capacity of the proteostasis network and was considered to be a key feature of the pathologic mechanisms of neurodegenerative diseases. Recent evidence suggests that the formation of intracellular aggregates is actually protective, sequestering potentially toxic misfolding proteins away from the cellular milieu. Evidence from yeast indicates that misfolding proteins are actively recognized by the proteostasis network and sequestered into distinct protein quality control compartments. Misfolding proteins, such as mutant Huntingtin, that form amyloid aggregates are directed to the insoluble protein deposit (IPOD) while non-amyloidogenic proteins, such as mutant SOD1, are transported to the juxtanuclear quality control compartment (JUNQ). However, the regulation of these pathways by the proteostasis network in mammalian cells remains poorly characterized. Our objective is to elucidate the mechanisms by which the proteostasis network in mammalian cells regulates the formation of IPOD and JUNQ to selectively sequester amyloid and non-amyloid aggregates (Specific Aim 1). Moreover, we will identify specific subnetworks of proteostasis factors that directly interact with misfolding proteins to direct them either to IPOD or JUNQ (Specific Aim 2). Finally, we will investigate the impact of disease-associated proteins and stress signaling on the function of these proteostasis factors that influence JUNQ and IPOD formation and function (Specific Aim 3). The characterization of these pathways in mammalian cells will provide new insights into the regulation of protein aggregation as it pertains to neurodegenerative disease. Furthermore, this strategy will identify novel targets for therapeutic intervention in a variety of diseases associated with protein misfolding and aggregation.